This invention relates to ribozymes, which are molecules containing catalytic RNA sequences that are capable of cleaving RNA sequences in a substrate.
U.S. Pat. No. 5,149,796 describes chimeric DNA-RNA-DNA-RNA-DNA ribozymes of formulas I and II:
3xe2x80x2 X-aaag-Y-aguaguc-Z 5xe2x80x2 (5xe2x80x2 Z-cugauga-Y-gaaa-X 3xe2x80x2)xe2x80x83xe2x80x83I.
3xe2x80x2 X-caaag-Y-aguaguc-Z 5xe2x80x2 (5xe2x80x2 Z-cugauga-Y-gaaac-X 3xe2x80x2)xe2x80x83xe2x80x83II.
in which X, Y and Z are DNA sequences and cugauga, gaaa and gaaac are catalytic RNA sequences. The flanking X and Z components may be any DNA sequences that allow base pairing with the substrate RNA at appropriate positions adjacent to the substrate cleavage site. Y may be any DNA sequence that base pairs inter se in the manner required for catalytic cleavage of the substrate by the RNA sequences.
The X and Z sequences may be substituted at the respective 3xe2x80x2 and 5xe2x80x2 ends with ligands to facilitate cell entry, targeting within the cell and ultimate stability of the catalysts.
The chimeric DNA-RNA-DNA-RNA-DNA ribozymes can be synthesized in known manner by commercially available DNA synthesizers.
The patent discloses specific chimeric ribozymes which are targeted to cleave HIV-1 RNA sequences and states that the chimeric ribozymes are administered by known delivery agents and systems such as liposomes, defective viral particles, viral capsids, and standard DNA/RNA transfective procedures.
This invention provides chimeric DNA-RNA-(Pr)n-RNA-DNA ribozymes which are capable of binding and cleaving an RNA substrate and which comprise the sequences of formula III or IV:
5xe2x80x2 Z-cugaugag-(Pr)n-cgaaa-X 3xe2x80x2xe2x80x83xe2x80x83III.
3xe2x80x2X-aaagc-(Pr)n-gaguaguc-Z-R-Z-cugaugag-(Pr)n-cgaaa-X 3xe2x80x2xe2x80x83xe2x80x83IV.
in which
X and Z comprise DNA sequences of at least six 2xe2x80x2-deoxy-ribonucleotide residues or modified 2xe2x80x2-deoxyribonucleotide residues that base pair with the RNA substrate at positions adjacent to the RNA cleavage site;
cugaugag and cgaaa are RNA sequences in which c, u, g and a are, respectively, residues of the ribonucleotides cytidylic acid, uridylic acid, guanylic and adenylic acid or, in the case of c and u, modified residues of c and u in which 2xe2x80x2-hydroxy is replaced with 2xe2x80x2-methoxy or other uncharged group such as 2xe2x80x2-allyloxy or 2xe2x80x2-fluoro;
Pr is a residue xe2x80x94P(O)(OH)xe2x80x94Oxe2x80x94CH2CH2CH2xe2x80x94Oxe2x80x94 derived from the C3 spacer reagent 3-(4,4xe2x80x2-dimethoxytrityloxy)propane-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;
n is at least 1, preferably 1-50, more preferably 2-10:
R is a residue xe2x80x94Oxe2x80x94CH2xe2x80x94C(CH2OH)(CH3)xe2x80x94CH2xe2x80x94Oxe2x80x94 derived from the bridging reagent 2-(dimethoxytrityl-O-methyl)-2-methyl-1,3-bis-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-propane.
Examples of modified deoxyribonucleotides which can be present in Z and X include phosphorothioates and methlyphosphonates. There is no upper limit on the length of Z and X, but they will generally be about 6-50 residues, preferably about 10-20 residues, more preferably about 12-15 residues.
Z and X can each be substituted at the respective 5xe2x80x2 and 3xe2x80x2 ends with ligands to facilitate cell entry, targeting within the cell, and stability of the ribozymes and to facilitate capture and detection in in vitro assays. Such ligands include other nucleotides, proteins, carbohydrates, lipids, steroid hormones, cholesterol, amino linkers, Pr spacers, dyes such as fluoroscein and rhodamine, capture reagents such as biotin, and others.
The ribozymes can be encapsulated into liposomes for delivery into cells in vitro or in vivo.
A specific ribozyme of the invention is:
5xe2x80x2AGCTTTATTcugaugag(Pr)4cgaaaGGCTTAAG3xe2x80x2xe2x80x83xe2x80x83(Seq. ID 1)
which contains 5xe2x80x2AGCTTTATTcugaugag3xe2x80x2 (SED ID NO. 1) and 5xe2x80x2cgaaaGGCTTAAG3xe2x80x2 (SEQ ID NO. 2)
where
c, u, g and a are, respectively, residues of the ribonucleotides cytidylic acid, uridylic acid, guanylic acid and adenylic acid;
C, T, G and A are, respectively, residues of the 2xe2x80x2-deoxyribonucleotides deoxycytidylic acid, deoxythymidylic acid, deoxyguanylic acid and deoxyadenylic acid
FIG. 1. The ribozyme containing SEQ ID NO. 1 and SEQ ID NO. 2 is targeted to cleave an HIV-1 RNA in the U5 region, as illustrated in FIG. 1.
Ribozymes of this invention can be designed and used for the same purposes and in the same ways that other ribozymes are used, as disclosed in U.S. Pat. No. 5,149,796, cited above, and as reviewed in Rossi, xe2x80x9cRibozymes, genomics and therapeutics,xe2x80x9d Chemistry and Biology, February 1999, vol. 6, no. 2, R33-R37, which are incorporated herein by reference. Thus, ribozymes of the invention can be used in the field of functional genomics as tools for studying gene functions and identifying potential new therapeutic targets for the treatment of disease. They can also be designed and used to treat RNA-mediated diseases such as HIV-1 infection or other diseases associated with altered expression or mutant forms of a gene or genes, including genetic diseases linked to allelic polymorphisms, and various cancers such as chronic myelogenous leukemia, breast cancer, and bladder cancer.
For example, use of a DNA-RNA chimeric ribozyme to study the pathogenetic role of the bcr-abl gene in Ph1+ leukemogenesis, and potentially to treat patients with Ph1+ chronic myelogenous leukemia, is disclosed in Snyder et al., xe2x80x9cRibozyme Inhibition of bcr-abl Gene Expression,xe2x80x9d Blood 82:2:600-605 (1992). As another example, a DNA-RNA chimeric ribozyme targeted to cleave mRNA for proliferating cell nuclear antigen (PCNA), and its use to reduce stent-induced stenosis in a porcine model, is disclosed in Frimerman et al., Circulation 99;697-703 (1999). As another example, a DNA-RNA chimeric ribozyme targeted to cleave mRNA for leukocyte-type 12 lipoxygenase (12-LO), and its use to study the specific effects of the 12-LO gene pathway in vascular disease, is disclosed in Gu et al., Circ. Res. 77:14-20 (1995). Ribozymes of this invention having the same RNA-binding flanking sequences as the ribozymes of the Snyder et al., Frimerman et al., and Gu et al. papers can be synthesized and used for the purposes disclosed in those papers. The disclosures of those papers are incorporated herein.
These examples are not intended to limit the invention. A ribozyme of the invention can be designed and synthesized to target any RNA which contains an NUH target sequence, where N is any ribonucleotide (C, U, G or A) and H is A, C or U, in an accessible binding site. Methods for identifying accessible binding sites in RNA are discussed in the Rossi review article cited above.